https://www.alphaknockout.com

Mouse Dlg2 Knockout Project (CRISPR/Cas9)

Objective: To create a Dlg2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dlg2 (NCBI Reference Sequence: NM_011807 ; Ensembl: ENSMUSG00000052572 ) is located on Mouse 7. 23 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 23 (Transcript: ENSMUST00000107196). Exon 5 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele display lower surface expression of NMDA receptor (NMDAR) subunits NR2A and NR2B in dorsal horn neurons and significantly reduced NMDAR-mediated excitatory synaptic currents and NMDAR-dependent persistent inflammatory or nerve injury-induced neuropathic pain.

Exon 5 starts from about 12.13% of the coding region. Exon 5 covers 4.89% of the coding region. The size of effective KO region: ~125 bp. The KO region does not have any other known gene.

Page 1 of 9 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 5 23

Legends Exon of mouse Dlg2 Knockout region

Page 2 of 9 https://www.alphaknockout.com

Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 5 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

Overview of the Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 5 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

Page 3 of 9 https://www.alphaknockout.com

Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(30.5% 610) | C(18.0% 360) | T(32.8% 656) | G(18.7% 374)

Note: The 2000 bp section upstream of Exon 5 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(34.6% 692) | C(15.2% 304) | T(29.0% 580) | G(21.2% 424)

Note: The 2000 bp section downstream of Exon 5 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

Page 4 of 9 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr7 + 91870377 91872376 2000 browser details YourSeq 112 8 254 2000 89.5% chr12 + 82023817 82024101 285 browser details YourSeq 98 1 113 2000 95.4% chr12 - 58848872 58849413 542 browser details YourSeq 94 8 182 2000 81.7% chr19 + 16117470 16117597 128 browser details YourSeq 93 1 111 2000 95.2% chr10 + 4165822 4165935 114 browser details YourSeq 92 8 125 2000 89.3% chr2 + 170164931 170165040 110 browser details YourSeq 90 1 97 2000 96.9% chr13 + 73423993 73424501 509 browser details YourSeq 90 1 97 2000 97.0% chr10 + 85175248 85175350 103 browser details YourSeq 89 1 97 2000 95.9% chr18 - 67799449 67799545 97 browser details YourSeq 89 1 97 2000 95.9% chr1 - 160134418 160134514 97 browser details YourSeq 89 1 95 2000 96.9% chr18 + 75005343 75005437 95 browser details YourSeq 89 7 124 2000 94.0% chr12 + 111753897 111754157 261 browser details YourSeq 88 1 96 2000 95.9% chr12 + 84154096 84154191 96 browser details YourSeq 87 1 99 2000 91.9% chr1 - 59088251 59088348 98 browser details YourSeq 84 5 96 2000 95.7% chr4 + 120443696 120443787 92 browser details YourSeq 84 9 98 2000 96.7% chr11 + 22046969 22047058 90 browser details YourSeq 83 1 92 2000 92.3% chr2 + 8954407 8954496 90 browser details YourSeq 83 8 96 2000 96.7% chr16 + 90404938 90405026 89 browser details YourSeq 83 1 93 2000 94.7% chr16 + 65508292 65508384 93 browser details YourSeq 83 1 97 2000 94.7% chr16 + 29691371 29691467 97

Note: The 2000 bp section upstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

BLAT Search Results (down)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr7 + 91872502 91874501 2000 browser details YourSeq 46 771 838 2000 92.8% chr18 - 63997215 63997405 191 browser details YourSeq 43 791 842 2000 92.0% chr9 - 36250945 36250996 52 browser details YourSeq 41 768 996 2000 60.0% chr9 - 20278263 20278338 76 browser details YourSeq 41 790 847 2000 85.5% chr10 - 56186792 56186846 55 browser details YourSeq 40 772 845 2000 81.3% chr5 - 133299313 133299383 71 browser details YourSeq 36 791 852 2000 72.1% chr7 - 116790613 116790661 49 browser details YourSeq 35 791 828 2000 97.3% chr1 - 176653187 176653225 39 browser details YourSeq 35 791 837 2000 81.6% chr19 + 51757553 51757595 43 browser details YourSeq 33 794 836 2000 94.5% chr2 + 133224911 133224960 50 browser details YourSeq 30 791 831 2000 73.6% chr10 - 88372437 88372470 34 browser details YourSeq 29 791 824 2000 94.0% chr1 - 100240769 100240803 35 browser details YourSeq 28 1892 1923 2000 93.8% chr15 - 7259228 7259259 32 browser details YourSeq 28 1954 1993 2000 71.0% chr10 + 84502505 84502535 31 browser details YourSeq 23 925 953 2000 89.7% chr1 + 179413346 179413374 29 browser details YourSeq 22 803 840 2000 79.0% chr3 - 150861049 150861086 38

Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 https://www.alphaknockout.com

Gene and information: Dlg2 discs large MAGUK scaffold protein 2 [ Mus musculus (house mouse) ] Gene ID: 23859, updated on 10-Oct-2019

Gene summary

Official Symbol Dlg2 provided by MGI Official Full Name discs large MAGUK scaffold protein 2 provided by MGI Primary source MGI:MGI:1344351 See related Ensembl:ENSMUSG00000052572 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Dlgh2; PSD93; Gm1197; Gm21505; A330103J02Rik; B230218P12Rik; B330007M19Rik Expression Biased expression in frontal lobe adult (RPKM 12.3), cortex adult (RPKM 11.4) and 5 other tissues See more Orthologs human all

Genomic context

Location: 7 E1; 7 51.07 cM See Dlg2 in Genome Data Viewer Exon count: 40

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (90476188..92449246)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (98239296..99597599)

Chromosome 7 - NC_000073.6

Page 6 of 9 https://www.alphaknockout.com

Transcript information: This gene has 22 transcripts

Gene: Dlg2 ENSMUSG00000052572

Description discs large MAGUK scaffold protein 2 [Source:MGI Symbol;Acc:MGI:1344351] Gene Synonyms A330103J02Rik, B230218P12Rik, B330007M19Rik, Chapsyn-110, Dlgh2, Gm21505, LOC382816, PSD93 Location Chromosome 7: 90,476,672-92,449,247 forward strand. GRCm38:CM001000.2 About this gene This gene has 22 transcripts (splice variants), 180 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dlg2-204 ENSMUST00000107196.9 7490 852aa ENSMUSP00000102814.2 Protein coding CCDS40021 Q91XM9 TSL:1 GENCODE basic APPRIS P2

Dlg2-202 ENSMUST00000098308.3 2329 481aa ENSMUSP00000095910.2 Protein coding CCDS57562 D3YWU0 TSL:1 GENCODE basic

Dlg2-217 ENSMUST00000231777.2 7801 994aa ENSMUSP00000155862.2 Protein coding - A0A338P6J0 GENCODE basic APPRIS ALT2

Dlg2-218 ENSMUST00000238467.1 7573 919aa ENSMUSP00000158695.1 Protein coding - - GENCODE basic

Dlg2-220 ENSMUST00000238619.1 7559 887aa ENSMUSP00000158731.1 Protein coding - - GENCODE basic

Dlg2-219 ENSMUST00000238608.1 7401 901aa ENSMUSP00000158616.1 Protein coding - - GENCODE basic

Dlg2-201 ENSMUST00000074273.9 5278 870aa ENSMUSP00000073885.3 Protein coding - E9Q2L2 TSL:5 GENCODE basic APPRIS ALT2

Dlg2-203 ENSMUST00000107193.7 4910 755aa ENSMUSP00000102811.1 Protein coding - D3YUZ8 TSL:5 GENCODE basic

Dlg2-222 ENSMUST00000239136.1 540 78aa ENSMUSP00000159135.1 Protein coding - - CDS 3' incomplete

Dlg2-215 ENSMUST00000208919.1 396 47aa ENSMUSP00000159178.1 Protein coding - - CDS 3' incomplete TSL:3

Dlg2-209 ENSMUST00000146563.8 4236 No protein - Retained intron - - TSL:5

Dlg2-210 ENSMUST00000152139.7 2934 No protein - Retained intron - - TSL:1

Dlg2-211 ENSMUST00000207095.1 2397 No protein - Retained intron - - TSL:NA

Dlg2-206 ENSMUST00000129818.1 2218 No protein - Retained intron - - TSL:1

Dlg2-221 ENSMUST00000238788.1 1216 No protein - Retained intron - - -

Dlg2-213 ENSMUST00000207891.1 448 No protein - Retained intron - - TSL:3

Dlg2-208 ENSMUST00000138389.7 3218 No protein - lncRNA - - TSL:1

Dlg2-216 ENSMUST00000209170.1 1057 No protein - lncRNA - - TSL:2

Dlg2-207 ENSMUST00000135581.2 964 No protein - lncRNA - - TSL:3

Dlg2-205 ENSMUST00000128592.2 922 No protein - lncRNA - - TSL:5

Dlg2-212 ENSMUST00000207798.1 842 No protein - lncRNA - - TSL:5

Dlg2-214 ENSMUST00000208377.1 672 No protein - lncRNA - - TSL:5

1.99 Mb Forward strand 90.5Mb 91.0Mb 91.5Mb 92.0Mb (Comprehensive set... Dlg2-211 >retained intron A930002H02Rik-201 >TEC Gm24552-201 >snRNA Gm44679-201 >TEC Gm45130-201 >TEC Dlg2-202 >protein coding

Dlg2-215 >protein coding Gm45159-201 >lncRNGAm45176-201 >TEC Dlg2-222 >protein coding Gm45129-201 >TEC Dlg2-206 >retained intron

Dlg2-216 >lncRNA Gm45178-201 >TEC Gm45183-201 >TEC Gm44675-201 >TEC Gm45131-201 >TEC

Dlg2-217 >protein coding Page 7 of 9

Gm45179-201 >TEC Gm44678-201 >TEC Gm44676-201 >TEC B230206I08Rik-201 >TEC

Dlg2-204 >protein coding

Dlg2-209 >retained intron

Dlg2-201 >protein coding

Gm45177-201 >TEC Dlg2-221 >retained intron Dlg2-212 >lncRNA

Gm45182-201 >TEC Gm44677-201 >TEC Dlg2-213 >retained intron

Gm44680-201 >TEC Gm45201-201 >TEC

Gm44681-201 >TEC Dlg2-208 >lncRNA

Dlg2-220 >protein coding

Dlg2-218 >protein coding

Dlg2-219 >protein coding

Dlg2-210 >retained intron Dlg2-207 >lncRNA

C030038I04Rik-201 >TEC Dlg2-214 >lncRNA

Dlg2-203 >protein coding

Dlg2-205 >lncRNA

Contigs Genes < Tmem126b-201protein coding< Gm45162-201processed pseudogene < Gm22080-201snRNA < Gm23928-201snRNA < 4931412I15Rik-201TEC (Comprehensive set...

< Tmem126b-202retained intron < Gm45161-201TEC < 4930567K12Rik-201lncRNA

< Gm27470-201misc RNA

< Gm27660-201misc RNA

Regulatory Build

90.5Mb 91.0Mb 91.5Mb 92.0Mb Reverse strand 1.99 Mb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

pseudogene processed transcript RNA gene 1.99 Mb Forward strand 90.5Mb 91.0Mb 91.5Mb 92.0Mb Genes (Comprehensive set... Dlg2-211 >retained intron A930002H02Rik-201 >TEC Gm24552-201 >snRNA Gm44679-201 >TEC Gm45130-201 >TEC Dlg2-202 >protein coding

Dlg2-215 >protein coding Gm45159-201 >lncRNGAm45176-201 >TEC Dlg2-222 >protein coding Gm45129-201 >TEC Dlg2-206 >retained intron

Dlg2-216 >lncRNA Gm45178-201 >TEC Gm45183-201 >TEC Gm44675-201 >TEC Gm45131-201 >TEC https://www.alphaknockout.com

Dlg2-217 >protein coding

Gm45179-201 >TEC Gm44678-201 >TEC Gm44676-201 >TEC B230206I08Rik-201 >TEC

Dlg2-204 >protein coding

Dlg2-209 >retained intron

Dlg2-201 >protein coding

Gm45177-201 >TEC Dlg2-221 >retained intron Dlg2-212 >lncRNA

Gm45182-201 >TEC Gm44677-201 >TEC Dlg2-213 >retained intron

Gm44680-201 >TEC Gm45201-201 >TEC

Gm44681-201 >TEC Dlg2-208 >lncRNA

Dlg2-220 >protein coding

Dlg2-218 >protein coding

Dlg2-219 >protein coding

Dlg2-210 >retained intron Dlg2-207 >lncRNA

C030038I04Rik-201 >TEC Dlg2-214 >lncRNA

Dlg2-203 >protein coding

Dlg2-205 >lncRNA

Contigs Genes < Tmem126b-201protein coding< Gm45162-201processed pseudogene < Gm22080-201snRNA < Gm23928-201snRNA < 4931412I15Rik-201TEC (Comprehensive set...

< Tmem126b-202retained intron < Gm45161-201TEC < 4930567K12Rik-201lncRNA

< Gm27470-201misc RNA

< Gm27660-201misc RNA

Regulatory Build

90.5Mb 91.0Mb 91.5Mb 92.0Mb Reverse strand 1.99 Mb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

pseudogene processed transcript RNA gene

Page 8 of 9 https://www.alphaknockout.com

Transcript: ENSMUST00000107196

1.36 Mb Forward strand

Dlg2-204 >protein coding

ENSMUSP00000102... PDB-ENSP mappings Low complexity (Seg) Coiled-coils (Ncoils) Superfamily PDZ superfamily P-loop containing nucleoside triphosphate hydrolase

SH3-like domain superfamily SMART Disks large homologue 1, N-terminal PEST domain SH3 domain Guanylate kinase/L-type calcium channel beta subunit

PDZ domain Pfam Disks large homologue 1, N-terminal PEST domain SH3 domain Guanylate kinase/L-type calcium channel beta subunit

PDZ-associated domain of NMDA receptors

PDZ domain PROSITE profiles PDZ domain SH3 domain Guanylate kinase-like domain

PROSITE patterns Guanylate kinase, conserved site

PIRSF Disks large 1-like PANTHER PTHR23122

PTHR23122:SF67 Gene3D 2.30.42.10 2.30.30.40

3.30.63.10

3.40.50.300 CDD cd00992 cd00071

Disks Large homologue 2, SH3 domain

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend

synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 852

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

Page 9 of 9